期刊
NANO LETTERS
卷 19, 期 11, 页码 8245-8249出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.nanolett.9b03736
关键词
Binding affinity; competitive binding; fluorescence correlation spectroscopy; fluorescence labeling; fluorescent probes; protein-protein interactions
类别
资金
- Volkswagen Foundation [91069]
- German Science Foundation [SFB 1177]
Fluorescence methods are important tools in modern biology. Direct labeling of biomolecules with a fluorophore might, however, change interaction surfaces. Here, we introduce a competitive binding assay in combination with fluorescence correlation spectroscopy that reports binding affinities of both labeled and unlabeled biomolecules to their binding target. We investigated how fluorophore labels at different positions of a DNA oligonucleotide affect hybridization to a complementary oligonucleotide and found dissociation constants varying within 2 orders of magnitude. We next demonstrated that placing a fluorophore label at position Leu280 in the protein ligand internalin B does not alter the binding affinity to the MET receptor tyrosine kinase, compared to unlabeled internalin B. Our approach is simple to implement and can be applied to investigate the influence of fluorophore labels in a large variety of biomolecular interactions.
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